WO2013093642A1 - Method for labeling intracellular and extracellular targets of leukocytes - Google Patents
Method for labeling intracellular and extracellular targets of leukocytes Download PDFInfo
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- WO2013093642A1 WO2013093642A1 PCT/IB2012/003071 IB2012003071W WO2013093642A1 WO 2013093642 A1 WO2013093642 A1 WO 2013093642A1 IB 2012003071 W IB2012003071 W IB 2012003071W WO 2013093642 A1 WO2013093642 A1 WO 2013093642A1
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- 238000000034 method Methods 0.000 title claims abstract description 75
- 210000000265 leukocyte Anatomy 0.000 title claims abstract description 41
- 230000003834 intracellular effect Effects 0.000 title claims abstract description 27
- 238000002372 labelling Methods 0.000 title claims abstract description 8
- 239000011230 binding agent Substances 0.000 claims description 33
- 239000003795 chemical substances by application Substances 0.000 claims description 24
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 21
- 239000000203 mixture Substances 0.000 claims description 19
- 210000004369 blood Anatomy 0.000 claims description 16
- 239000008280 blood Substances 0.000 claims description 16
- 230000001413 cellular effect Effects 0.000 claims description 13
- 239000003599 detergent Substances 0.000 claims description 11
- 210000003743 erythrocyte Anatomy 0.000 claims description 11
- 150000001875 compounds Chemical class 0.000 claims description 10
- 230000003472 neutralizing effect Effects 0.000 claims description 10
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 9
- 238000005119 centrifugation Methods 0.000 claims description 8
- 238000000684 flow cytometry Methods 0.000 claims description 8
- 238000004132 cross linking Methods 0.000 claims description 7
- 230000000694 effects Effects 0.000 claims description 6
- KSAVQLQVUXSOCR-UHFFFAOYSA-M sodium lauroyl sarcosinate Chemical compound [Na+].CCCCCCCCCCCC(=O)N(C)CC([O-])=O KSAVQLQVUXSOCR-UHFFFAOYSA-M 0.000 claims description 6
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 5
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 5
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 claims description 5
- 229920002866 paraformaldehyde Polymers 0.000 claims description 5
- 235000019270 ammonium chloride Nutrition 0.000 claims description 4
- 230000002934 lysing effect Effects 0.000 claims description 4
- 102000004895 Lipoproteins Human genes 0.000 claims description 3
- 108090001030 Lipoproteins Proteins 0.000 claims description 3
- 150000001412 amines Chemical class 0.000 claims description 3
- 230000009089 cytolysis Effects 0.000 claims description 3
- 102000039446 nucleic acids Human genes 0.000 claims description 3
- 108020004707 nucleic acids Proteins 0.000 claims description 3
- 150000007523 nucleic acids Chemical class 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 150000003242 quaternary ammonium salts Chemical class 0.000 claims description 3
- 210000003567 ascitic fluid Anatomy 0.000 claims description 2
- 210000001185 bone marrow Anatomy 0.000 claims description 2
- 239000000243 solution Substances 0.000 description 65
- 210000004027 cell Anatomy 0.000 description 23
- 238000010186 staining Methods 0.000 description 21
- 238000005406 washing Methods 0.000 description 18
- 241000976924 Inca Species 0.000 description 14
- 239000000523 sample Substances 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000000834 fixative Substances 0.000 description 4
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 238000010212 intracellular staining Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 150000001335 aliphatic alkanes Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 229920001993 poloxamer 188 Polymers 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 1
- 229940100484 5-chloro-2-methyl-4-isothiazolin-3-one Drugs 0.000 description 1
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 1
- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 229920001400 block copolymer Polymers 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- DHNRXBZYEKSXIM-UHFFFAOYSA-N chloromethylisothiazolinone Chemical compound CN1SC(Cl)=CC1=O DHNRXBZYEKSXIM-UHFFFAOYSA-N 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- HQPMKSGTIOYHJT-UHFFFAOYSA-N ethane-1,2-diol;propane-1,2-diol Chemical compound OCCO.CC(O)CO HQPMKSGTIOYHJT-UHFFFAOYSA-N 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- 229920002503 polyoxyethylene-polyoxypropylene Polymers 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 208000019585 progressive encephalomyelitis with rigidity and myoclonus Diseases 0.000 description 1
- 239000013074 reference sample Substances 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
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- 239000000725 suspension Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
- G01N33/56972—White blood cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5094—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for blood cell populations
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/70514—CD4
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70589—CD45
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- G01N2333/91205—Phosphotransferases in general
Definitions
- the present invention relates to methods for labeling intracellular and extracellular targets of leukocytes, as well as to kits for performing said methods.
- mAbs monoclonal antibodies
- the surface markers of the cells are stained first, followed by one ore more washing step(s) (i.e. centrifuging the cells, discarding the supernatant, and then resuspending them in fresh buffer), in order to remove any unbound binding agent. Then, the cells are fixed, again followed by one or more washing steps to remove the fixative, permeabilized, washed again, then stained with the intracellular binding agents and finally washed again.
- washing step(s) i.e. centrifuging the cells, discarding the supernatant, and then resuspending them in fresh buffer
- the technical problem underlying the present invention is to provide an improved method for the labeling of intracellular and extracellular targets of leukocytes that is fast and not very labor intensive, does avoid any centrifugation steps, allows the use of binding agent cocktails and can be easily automated.
- the present invention relates to a method for labeling intracellular and extracellular targets of leukocytes, comprising the steps of:
- a cellular composition comprising at least leukocytes and red blood cells
- a first solution comprising one or more agent(s) that are capable of cross-linking intracellular proteins, lipoproteins and nucleic acids of said leukocytes;
- a second solution comprising one or more agent(s) that are capable of permeabilizing said leukocytes, lysing said red blood cells and neutralizing the cross-linking activity of said one or more agent(s) in said first solution, characterized in that the added volume of said second solution is between 1 and 10 times the total volume of said combination;
- each of said binding agents comprises a detectable agent
- the method of the present invention is referred to herein as INCA method (Intracellular No Centrifuge Assay).
- the cellular composition containing the leukocytes to be stained is a biological fluid containing cells in suspension.
- the cellular composition is selected from the group consisting of whole blood, bone marrow, peritoneal fluid, cephalic fluids, dissociated lymph nodes and other dissociated tissues.
- the cellular composition is whole blood.
- the combination obtained in step (a) of the method of the present invention is incubated for 5 to 20 minutes, in another embodiment for 10 to 15 minutes, prior to step (b).
- the one or more agent(s) that are capable of cross-linking intracellular proteins, lipoproteins and nucleic acids of leukocytes used in step (a) are, according to one embodiment, selected from the group consisting of formaldehyde, paraformaldehyde, and glutaraldehyde.
- said agent(s) are contained in said combination in an amount of 0.5% to 2% (v/v) after addition of the first solution, and in said first solution in an amount of between 5% and 15% (v/v).
- step (a) of the method of the present invention is performed for fixing the cells contained in the cellular composition.
- the second solution added in step (b) of the method of the present invention comprising one or more agent(s) that are capable of permeabilizing said leukocytes, lysing said red blood cells and neutralizing the cross-linking activity of said one or more agent(s) in said first solution, comprises (i) a detergent in an amount that is adapted to effect the lysis of substantially all red blood cells contained in the cellular composition, while the majority of leukocytes contained in the cellular composition are not lysed, and (ii) a neutralizing agent for neutralizing said agent(s) of step (a).
- said detergent contains a C 12 -type alkane residue, according to another embodiment sodium N-lauroyl sarcosine, wherein the detergent is, according to one embodiment, contained in said second solution in an amount of 0.05% to 0.5% (w/v), according to another embodiment in an amount of 0.1 % to 0.3% (w/v).
- said neutralizing agent is a quaternary ammonium salt or an amine containing compound, according to another embodiment a compound, selected from the group consisting of ammonium chloride (NH 4 CI), glycine, tris(hydroxymethyl)aminomethane (Tris), and ethanolamine, wherein in a still further embodiment it is ammonium chloride.
- said neutralizing agent is contained in said second solution in a concentration of 1 to 100 mM, in another embodiment in a concentration of 5 to 20 mM.
- said second solution has a pH of 6.5 or lower.
- the added volume of said second solution added in step (b) of the method of the present invention is between 1 and 10 times, in another embodiment between 4 and 8 times, and in a still further embodiment 6 times the total volume of the combination obtained in step (a).
- step (b) of the method of the present invention is performed for permeabilizing the leukocytes and lysing the red blood cells contained in the cellular composition, as well as for neutralizing the cross-linking activity of said one or more agent(s) in said first solution added in step (a).
- Step (c) of the method of the present invention can be performed concurrently with step (b).
- the binding agents added in step (c) can already be contained in the second solution added in step (b).
- said binding agents can be added subsequently to addition of said second solution, wherein the extracellular and intracellular binding agents can be added either together as a binding agent cocktail, or separately.
- the binding agents are molecular stains, in another embodiment antibodies, in a still further embodiment monoclonal antibodies.
- the detectable agent comprised in said binding agents is not particularly limited, wherein suitable detectable agents are known in the art.
- said detectable agent is selected from the group consisting of a biotin, an enzyme, and a fluorescent moiety or compound, wherein, according to another embodiment, it is a fluorescent moiety or compound.
- the combination obtained in step (c) is incubated for 15 to 60 minutes, in another embodiment for 30 to 45 minutes, prior to step (d).
- step (c) of the method of the present invention is performed for staining the leukocytes contained in the cellular composition.
- the third solution added to said combination, i.e. to the combination obtained in step (c), in step (d) of the method of the present invention comprises according to one embodiment (i) a fixative, which in one embodiment is selected from the group consisting of formaldehyde, paraformaldehyde, and glutaraldehyde, and (ii) a detergent, containing, according to one embodiment, a C 12 -type alkane residue, according to another embodiment sodium N-lauroyl sarcosine.
- the above fixative is contained in the third solution in an amount of between 0.01 % and 1 % (v/v), and the above detergent in an amount of 0.01 % to 0.5% (w/v).
- the third solution further comprises a compound, selected from the group consisting of dextrane sulfate and Pluronic F-68, which is a polyoxyethylene-polyoxypropylene block copolymer with the linear formula (C 3 H 6 O.C 2 H O) x .
- Said further compound is, according to one embodiment, contained in the third solution in an amount of 0.1 % (w/v).
- the method of the present invention does not contain any centrifugation step, e.g. for purifying said leukocytes, prior to step (d) or after step (b) or step (c), or any additional washing steps.
- the method of the present invention comprises one washing step after step (d), i.e. a step of centrifuging the cells, discarding the supernatant, and resuspending the cells in a suitable volume of the third solution used in step (d). This washing step can improve the signal-to-noise ratio which is particularly useful when working with rather dim stainings, and further concentrates the cells.
- the method of the present invention further comprises the step of detecting the bound binding agents on the leukocytes, according to one embodiment by flow cytometry.
- all method steps of the method of the present invention are performed at room temperature.
- the method of the present invention is an automated method.
- the present invention relates to a kit for performing the methods of the present invention, comprising a first solution, a second solution, and a third solution, wherein said first, second, and third solutions are as defined above.
- the kit of the present invention comprises:
- kit of the present invention further comprises:
- binding agent comprising a label that is detectable by flow cytometry, wherein said binding agent specifically binds to an extracellular target of leukocytes
- binding agent comprising a label that is detectable by flow cytometry, wherein said binding agent specifically binds to an intracellular target of leukocytes
- extra- and intracellular binding agents are provided in separate tubes or as components of said second solution, and
- binding agents and the respective detectable labels are as defined above.
- the above first, second, and third solutions can further contain suitable additional components in suitable concentrations, such as buffer substances, e.g. 2-(N- morpholino)ethanesulfonic acid (MES) or phosphate-buffered saline (PBS); salts, e.g. sodium chloride; serum or serum components, e.g. bovine serum albumin; and preservatives, e.g. Proclin (5-chloro-2-methyl-4-isothiazolin-3- one).
- buffer substances e.g. 2-(N- morpholino)ethanesulfonic acid (MES) or phosphate-buffered saline (PBS)
- salts e.g. sodium chloride
- serum or serum components e.g. bovine serum albumin
- preservatives e.g. Proclin (5-chloro-2-methyl-4-isothiazolin-3- one).
- the present invention advantageously provides an improved method for labeling intracellular and extracellular targets of leukocytes, as well as respective kits for performing said method.
- all time- and labor-consuming centrifugation steps can be omitted.
- This significantly speeds up the procedure and in addition protects the structure of the cells, so that e.g. light scatter properties are improved as compared to centrifuged cells.
- a soft fixation step maintains the surface structures of the leukocytes intact, and remaining fixative is inactivated during permeabilization, so that the cells can be stained with extracellular binding agents even after fixation.
- the method of the present invention enables the use of cocktails comprising respective mixes of extra- and intracellular binding agents.
- Solutions 1 , 2 and 3 as well as the employed method correspond to example 4.
- Figure 2 shows the respective stainings with the antibody (FL2) versus sideward scatter.
- the methods according to the present invention stained equal amounts of cells compared to the prior art method and showed a clearer separation from the unstained cells with an RMFI (relative mean fluorescence intensity, which is a measure for the signal-to-noise ration) of 30 (INCA wash) and 15 (INCA no wash).
- RMFI relative mean fluorescence intensity, which is a measure for the signal-to-noise ration
- FIG. 3 shows the respective stainings with the anti-CD4 antibody (FL1 ) versus the intracellular stainings with the anti-FoxP3 antibody (FL4).
- the method known in the art stained only a small population of 0.36% of the cells, whereas the method of the present invention containing an additional washing step stained a substantial population of 4.3%.
- Solution 1 Formaldehyde 5.5% wt/vol Na2HP04 3mM
- a human whole blood sample (WBS) was processed in the following manner.
- solution 1 is called R1
- solution 2 is called R2
- solution 3 is called R3.
- the following table 3 provides signal/noise values with regard to 3 intracellular targets and 2 extracellular targets.
- the conjugates used were Anti-MPO-FITC, anti-CD79a-PE, anti-CD3-ECD (IOTest3 cocktail PN IM3464U), and Anti-CD14- PC7 (PN A22331 ).
- the present invention provides a robust system and method for detecting intra- and extracellular epitopes which works well over a great variety of buffer compositions including various concentrations of the buffer ingredients.
- Example 4 Influence of an additional washing step
- the conditions of Example 4 were varied insofar as a further washing step was added.
- solutions 1 , 2, and 3 as outlined in Example 4, above, were prepared and the following method for staining a human whole blood sample (WBS) with labeled antibodies was carried out.
- WBS human whole blood sample
- Example 4 the method for staining a human whole blood sample as outlined in Example 4 was carried out using the variations of solutions 1 , 2, and 3 as outline for reference experiment No. 1 given in Table 2 of Example 4a. In particular, it was determined as to whether the signal to noise ratio is drastically influenced by a variation of the pH of solution 2.
- the staining of the human whole blood sample was done by using a PE-labeled anti-ZAP-70 antibody.
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Abstract
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Priority Applications (10)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14/366,024 US9678073B2 (en) | 2011-12-21 | 2012-12-21 | Method for labeling intracellular and extracellular targets of leukocytes |
JP2014548248A JP6053818B2 (en) | 2011-12-21 | 2012-12-21 | Method for labeling intracellular and extracellular targets of leukocytes |
KR1020147016948A KR102067742B1 (en) | 2011-12-21 | 2012-12-21 | Method for labeling intracellular and extracellular targets of leukocytes |
BR112014015204-7A BR112014015204B1 (en) | 2011-12-21 | 2012-12-21 | METHOD FOR THE MARKING OF INTRACELLULAR AND EXTRACELLULAR TARGETS OF LEUKOCYTES |
CN201280063544.1A CN104011542B (en) | 2011-12-21 | 2012-12-21 | For marking the method for target and extracellular targets in leukocytic cell |
CA2859605A CA2859605C (en) | 2011-12-21 | 2012-12-21 | Method for labeling intracellular and extracellular targets of leukocytes |
AU2012356324A AU2012356324A1 (en) | 2011-12-21 | 2012-12-21 | Method for labeling intracellular and extracellular targets of leukocytes |
MX2014007284A MX352837B (en) | 2011-12-21 | 2012-12-21 | Method for labeling intracellular and extracellular targets of leukocytes. |
US15/188,856 US11635433B2 (en) | 2011-12-21 | 2016-06-21 | Method for labeling intracellular and extracellular targets of leukocytes |
AU2018247279A AU2018247279B2 (en) | 2011-12-21 | 2018-10-11 | Method For Labeling Intracellular And Extracellular Targets Of Leukocytes |
Applications Claiming Priority (2)
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US11635433B2 (en) | 2011-12-21 | 2023-04-25 | Beckman Coulter, Inc. | Method for labeling intracellular and extracellular targets of leukocytes |
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WO2017141789A1 (en) * | 2016-02-19 | 2017-08-24 | コニカミノルタ株式会社 | Method for storing blood-derived specimens and method for determining rare cells |
WO2019118355A1 (en) * | 2017-12-12 | 2019-06-20 | 10X Genomics, Inc. | Systems and methods for single cell processing |
SG11202011500UA (en) * | 2018-07-12 | 2020-12-30 | Beckman Coulter Inc | Fixable viability dyes and their uses |
CN111807809B (en) * | 2019-04-10 | 2021-12-28 | 盐城工业职业技术学院 | Preparation method of palm nanofiber-graphene-carbon nanotube composite aerogel |
CN113884671A (en) * | 2021-09-27 | 2022-01-04 | 苏州东岭生物技术有限公司 | Flow type dyeing kit and configuration method and application method thereof |
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EP2607899B1 (en) | 2016-08-10 |
JP2015500999A (en) | 2015-01-08 |
BR112014015204A2 (en) | 2017-06-13 |
US9678073B2 (en) | 2017-06-13 |
KR102067742B1 (en) | 2020-01-20 |
EP3104177A1 (en) | 2016-12-14 |
JP6053818B2 (en) | 2016-12-27 |
US11635433B2 (en) | 2023-04-25 |
CN104011542A (en) | 2014-08-27 |
US20160299139A1 (en) | 2016-10-13 |
CN104011542B (en) | 2016-04-27 |
BR112014015204B1 (en) | 2021-03-30 |
MX2014007284A (en) | 2015-10-29 |
CA2859605A1 (en) | 2013-06-27 |
US20150010923A1 (en) | 2015-01-08 |
MX352837B (en) | 2017-12-11 |
KR20140100536A (en) | 2014-08-14 |
CA2859605C (en) | 2020-07-07 |
AU2018247279A1 (en) | 2018-11-01 |
BR112014015204A8 (en) | 2017-06-13 |
AU2012356324A1 (en) | 2014-07-03 |
AU2018247279B2 (en) | 2020-12-24 |
EP2607899A1 (en) | 2013-06-26 |
US20230288416A1 (en) | 2023-09-14 |
EP3104177B1 (en) | 2020-11-25 |
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